The Impact Study Of TLR2on Keratinocytes Co-incubation With Different Dermatophytes

ObjectiveResearching the growth inhibition of keratinocytes co-incubation with Trichophyton rubrum、Microsporum canis and Microsporum gypseum. And observe the variation tendency of co-incubation after closed TLR2.MethodsIn5%CO2,37℃environment, keratinocytes cultured in DMEM medium containing10%fetal bovine were incubated, to be divided into two groups when the cells were grown to about80%. The stimulation group (group A) is that with serum-free medium configuration wire suspension (suspension concentration of4x105/ml) to replace the original culture medium. First admoveaturing the TLR2antibody (antibody concentration20ug/ml) to keratinocytes culture flask, and incubated at37℃for1hour to to blocking the role of TLR2before add to fungus suspension, that’s the the blocking group (group B). At the same time the blank group is established. By trypan blue counting method calculated the rate of fungal growth inhibition at different time points (4h,8h,16h and24h).ResultsThe result that keratinocytes were incubated with mycelium suspension at different time can be seen as follows:The stimulation group of Trichophyton rubrum growth inhibition is starting to show at4h, and16h reached a peak (67.8%±3.1%). Growth inhibition of blocking group closed TLR2obviously become weakened. Before and after the closure of the growth inhibition of the difference was statistically significant (P<0.05). The stimulation group of canis4h’s growth inhibition rate was41.8%±2.5%. As time lapse, growth inhibition percentage is70.0%±6.8%at16h and24h decreased gradually. After closed the TLR2, blocking group growth inhibition decreased significantly in the beginning of the4h. Growth inhibition before and after blocking TLR2was statistically significant (P <0.05). Stimulation group of plaster-like spores growth inhibition as times lasting constant increasing. After closed TLR2, blocking group growth inhibition rate of4h was15.6%±1.7%. And at each time point inhibition are weakened, the difference was statistically significant (P<0.05) before and after blocking.ConclusionKeratinocyte has an growth inhibition effect on Trichophyton rubrum, Microsporum canis and Microsporum gypseum when they incubated together. TLR2have an important impact in the inhibition of the process of mentioned above. ObjectiveObserving inflammatory cytokines IL-6, IL-8and TNF-a expression changes after richophyton rubrum, Microsporum canis and plaster-like spores co-incubation keratinocyte. As well as TLR2has been blocked. Investigating the role of TLR2in dermatophyte infection.MethodsUse the three dermatophytes mentioned above stimulated keratinocytes, and direct stimulation of keratinocyte is stimulation group (group A). Afeter closed TLR2is blocking group (Group B). The concentration of IL-6, IL-8and TNF-alpha have been detected by using ELISA at different time points. Before and after blocked TLR2there is a comparing of changes in the concentration of various cytokines. At last, blank group has needed.ResultsWhen Trichophyton rubrum stimulate keratinocytes IL-8concentration was significantly higher at each time point. Concentration of IL-8compared with the control group and the blocking group in each time point have difference and that’s statistically significant (P<0.05). Stimulated with IL-6and TNF-alpha concentration significantly increased more and more obviously as times flies. With time of24h respectively arrive (498.133±20.159) pg/ml and (32.133±3.674) pg/ml. The difference was statistically significant (P<0.05) compared wite the blank group. When TLR2blocked, the concentration of cytokines decreased and at8h,16h and24h the difference was statistically significant (P<0.05).IL-8dynamic expression were gradually increased after Microsporum canis stimulate keratinocytes. The concentration of4h can be achieved (542.867±37.846) pg/ml and24h increased to (671.358±26.247) pg/ml. At blocking TLR2each time point, cytokines concentration expression decreased. Compared each group the difference was statistically significant (P<0.05). There is no change of concentration when at4h and8h of stimulation group’s IL-6and TNF-α. But after16hours cytokine amount secretion increased significantly and gradually increased over time. At each time point IL-6, and TNF-alpha expression is reduced after blocking TLR2. There are statistically significant (P<0.05) between each groups at16h and24h.IL-8concentrations began to increase at the beginning of the Microsporum gypseum stimulate keratinocytes. Between the control group and the blocking group differences were statistically significant (P<0.05). Stimulation of IL-6dynamic expression levels has difference at different time points. And at4h and8h stimulation group and control group, stimulation group and the blocking group was no significant difference (P>0.05). The comparing between each groups has shown statistical difference (P<0.05). IL-6 concentration can respectively achieve (38.70±8.41) pg/ml and (51.70±10.32) pg/ml at16h and24h. TNF-alpha concentration of stimulated group began to increased significantly at16h,24h could up to (40.61±5.94)pg/m. The difference was statistically significant (P<0.05) between every two groups.ConclusionIL-6, IL-8and TNF-alpha amount that keratinocyte expressed were markedly increased when Trichophyton rubrum, Microsporum canis and Microsporum gypseum were stimulated keratinocytes. While cytokines expression decreased after blocking TLR2. In word, TLR2in keratinocytes anti-different skin Trichophyton infection has played an important regulatory role of the process that keratinocytes make IL-6, IL-8and TNF-alpha.